Hypersensitivity of visceral organs is a main characteristic of numerous functional digestive disorders including irritable bowel syndrome (IBS): a debilitating disorder that affects up to 15% of the US population and one that continues to present a vexing clinical challenge. Despite the large body of scientific studies that have examined its mechanisms, visceral hypersensitivity (VH) evolves in many cases into a chronic condition recalcitrant to conventional treatment. To better understand the mechanisms of chronic VH, we developed an animal model in which rats exhibit symptoms of chronic VH and neuronal hyperexcitability in the absence of obvious colon inflammation. The model is induced by colon irritation (CI) in neonatal rats and results in a consequent symptomatology that carries into adulthood. Using rats with CI, we recently noted a novel host immune response, characterized by activation of macrophage-like cells in the spinal cord called microglia. Inhibiting microglial activation by systemic administration of minocycline or by intrathecal application of TNP- ATP to block P2X4 receptors (ligand-gated cation channels activated by ATP and expressed in the spinal cord exclusively by hyperactive microglia) reversed VH in adult rats treated with neonatal CI. Our observation is intriguing on two levels: 1) it demonstrates microglial activation several months following neonatal colonic insult (at postnatal day PND 120), and 2) it reveals, for the first time, evidence for a microglial role in visceral pain. Therefore, in this proposal we hypothesize that hyperactive microglia contribute to chronic VH and neuronal hyperexcitability in the spinal cord. First, we propose to establish a correlation between VH and a hyperactive phenotype of microglia in rats with CI by identifying molecular targets selective for hyperactive microglia, and second, we propose to reverse neuronal hyperexcitability and chronic VH by suppressing microglial activation using minocycline or by selectively blocking phosphorylated-p38 and P2X4 receptors, two targets specifically expressed on hyperactive microglia. These studies will help expand the targets of pharmacological intervention to non-neuronal sites within the spinal cord, and improve the analgesic potency of biologic therapies, while minimizing undesirable side effects. PUBLIC HEALTH RELEVANCE Visceral pain is a hallmark of many gastroenterological ailments such as irritable bowel syndrome. It may be caused by acute inflammation of an internal organ or be residual to previous injury;its consequences can be debilitating to the patients and to their families and frustrating to the treating clinician. To ameliorate treatment options and renew the hope of many patients with intractable visceral pain, we show in this application preliminary evidence for successful reversal of pain behavior in rats with chronic visceral pain by blocking novel cellular and molecular targets in the spinal cord. Unique features of our proposal include the remote location of these targets from the site of the initial injury, the interdisciplinary approach, and the diversified expertise of our team of investigators.